TRANSCRIPTION a) Transcription in prokaryotes b ...libvolume1.xyz/.../inhibitorsofprokaryotictranscriptiontutorial2.pdf2 Fig. Stryer Transcription in prokaryotes and eukaryotes The

WS 13/14

Strukturbiologie, Transcription 1

TRANSCRIPTION

a) Transcription in prokaryotes

b) Transcription in eukaryotes

2

Fig.

Stryer

Transcription in prokaryotes and eukaryotes

The fundamental mechanism of transcription is conserved among cellular RNA

polymerases, yet there are also marked differences between prokaryotes and eukaryotes:

Transcription and translation are coupled in bacteria; wherease transcription and

translation are uncoupled in eukarya.

-Stages of Transcription: Initiation, Elongation, Termination

-“transcription bubble,” unwound region of about 15 base pairs of the DNA template and

some eight residues of the RNA transcript hybridized with the DNA in the center of the

bubble.

3

Transcription cycle in prokaryotes

INITIATION of transcription

1. Binding of polymerase as a

holoenzyme (s factor plus core

polymerase)

2. Open complex formation

(transcription “bubble”).

Unwinding of DNA, forming single

strandedness within the active site.

3. Initial RNA synthesis. Up to 10

bp of RNA is synthesized. During

this initial step the polymerase is

not very efficient and can easily fall

off. www.bmb.psu.edu/courses/bmmb501/bmmb597a_fa

03/reese/16_lect_gene_reg_1_.pdf -

3 distinct phases:

INITIATION,

ELONGATION,

TERMINATION.

4

Transcription cycle in prokaryotes

4. Release of the s factor and

synthesis beyond 10 bp of RNA

(transition from initiation to

elongation Durniak, K.J., Bailey, S.,

Steitz, T.A. (2008) The structure of a

transcribing T7 RNA polymerase in transition

from initiation to elongation Science). A

structural change in the polymerase

occurs. The “jaws” tighten down on

the DNA and the elongation

complex is MUCH more stable.

5. Highly processive ELONGATION

phase

6. TERMINATION. A termination

signal in the DNA forms a RNA

hairpin in the emerging transcript.

Its binding to polymerase stimulates

a change in conformation (opening

of the “jaws”) and release.

www.bmb.psu.edu/courses/bmmb501/bmmb597a_fa

03/reese/16_lect_gene_reg_1_.pdf -


a) Transcription in prokaryotes

In prokaryotes, transcription and translation are closely coupled.

Gene transcription is regulated by protein transcription factors that bind to

operator DNA and thus influence the ability of RNA polymerase to bind to

a promoter region and initiate transcription.

Protein transcription factors are regulated by cellular environmental factors

(e.g. transcription factors, allosteric effectors), which can include small

molecules, another protein or metal ions. Transcription can be blocked by

binding of a specific repressor (e.g. lac) protein at a DNA site called an

operator. These DNA binding proteins recognize specific DNA sequences via

distinct DNA-binding domains.

Gene transcription in bacteria, Schreiter, 2007


Transcription in prokaryotes - RNAP

Y.W. Yin and T.A. Steitz, Structural basis for the transition from initiation to

elongation transcription in T7 RNA polymerase. Science 298 (2002).


Transcription in prokaryotes

Crystal structures of the T.

thermophilus elongation

complex (ttEC) with the

non-hydrolysable

substrate analogue

AMPcPP (Vassylyev et al,

nature 2007, 3Å)

Overall view of the ttEC/AMPcPP

complex.

The DNA template, non-template and

RNA strands are in red, blue and yellow,

respectively.

The BH, the TH and the rest of the RNAP

molecule are in magenta, cyan and

grey, respectively.

The insertion and preinsertion NTP

analogues and Stl are designated by

green, orange and black, respectively.

The catalytic Mg2+ ions (MgI and MgII)

are shown as magenta spheres. a, b,

9

-Eukaryotes: Transcription and Translation are uncoupled

b) Transcription in eukaryotes

-Eukaryotes: 3 different

RNA polymerases (Pol I,

Pol II, Pol III):

Regulatory elements of

eukaryotic transcription

(TATA-box, -25)


3D structure of the nucleosome

Surface representation of the histone

octamer

Structure of the nucleosome core

particle; (14 independent DNA-binding

locations)

Review, Karolin Luger

-In eukaryotes: Chromatin is composed of nucleosomes, which consist of an

octamer of histones around which 147 base pairs of DNA are wrapped.

Structure of the nucleosome, T. Richmond Lab, 1997


Transcription in eukaryotes

4 October 2006

The Royal Swedish Academy of Sciences has decided to

award the Nobel Prize in Chemistry for 2006 to

Roger D. Kornberg

Stanford University, CA, USA

"for his studies of the molecular basis of eukaryotic

transcription".

Kornberg's contribution has culminated in his creation of detailed

crystallographic pictures describing the transcription apparatus in full

action in a eukaryotic cell. In his pictures (all of them created since 2000)

we can see the new RNA-strand gradually developing, as well as the role of

several other molecules necessary for the transcription process. The pictures

are so detailed that separate atoms can be distinguished and this makes it

possible to understand the mechanisms of transcription and how it is

regulated.


Structure determination of RNA

polymerase II and complexes

1983 2-D protein crystals on lipid layers

1991 2-D crystals seed 3-D crystals (poor diffraction-work under

Argon)

1998 Diffraction phased with heavy atom clusters

2000 Structure of RNA polII at 2.8 Å resolution

2002 Structure of transcribing complex 3.3 Å

2002-ongoing Series of structures of transcribing complexs (2.9-

4.4 Å), complexes with bound inhibitors .....

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The Pol II transcription machinery (>3 MioDa)

Pol II is capable of unwinding DNA, synthesizing RNA, and rewinding

DNA. But Pol II alone is incapable of recognizing a promoter and

initiating transcription. For these essential functions, the participation

of the General Transcription Factors is required. Mediator is co-

activator, a co-repressor, and a general transcription factor all in one.

Mediator, a megaDalton multiprotein complex, enables the regulation

of transcription; it bridges between gene activator proteins at

enhancers and RNA polymerase II (pol II) at promoters.

Pol II: DNA unwinding

RNA polymerization

proofreading

GTFs (TFIIB,-D,E,F,-H): promoter recognition

Mediator: interaction with activator proteins

and polII; essential for transcription


Structure determination of the polymerase in

the form of a transcribing complex (3.3Å)

(B) Comparison of structures of

free Pol II (top) and the Pol II

transcribing complex (bottom). The

clamp (yellow) closes on DNA

and RNA, which are bound in the

cleft above the active center. The

remainder of the protein is in gray.

16

DNA can be seen entering the

transcribing complex in duplex

form and unwinding three bases

before the active site. Then the

template strand makes a sharp

bend, and as a result, the next

base is flipped, pointing down

towards the active site. This base

is paired with that of the

ribonucleotide just added to the

RNA strand. The structure reveals

eight more DNA-RNA hybrid base

pairs and one additional base on

the template DNA strand. The

remainder of the template strand,

the RNA, and the nontemplate

DNA strand are not seen, due to

motion or disorder.

Crystal structure of the Pol II transcribing complex

Gnatt et al, Science 2001


Transcription Initiation mechanisms

How is straight duplex promoter DNA melted, bent, and inserted in the

Pol II active center, enabling the initiation of transcription?

Bushnell, D.A., et al. (2004) Structural basis of transcription: an RNA polymerase II-TFIIB

cocrystal at 4.5 Ångströms. Science

These DNA

transactions are

made by the GTF’s

TFIIB , -D, -E, -F, and

-H.

TFIIB stabilizes an

initial transcribing

complex and the N-

terminal region

forms Zn ribbon and

B finger.

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Transcription Initiation mechanisms

The structure of the Pol II-TFIIB complex revealed distinct functions of

the N- and C-terminal domains of TFIIB. The N-terminal domain

(yellow) begins with a Zn ribbon that binds the Pol II surface adjacent

to the clamp and wall.Then the polypeptide continues across the

saddle between the clamp and wall and plunges towards the active

center, from which it loops back and remerges across the saddle.

19

The loop, termed the B finger, occupies almost the same location

as the DNA-RNA hybrid in a transcribing complex.

Superimposing the B finger and the DNA-RNA hybrid from the

transcribing complex structure reveals no interference with the

template DNA strand or with the RNA up to position 5, but a steric

clash with the RNA at positions 6 and beyond.


B finger is not only compatible

with a hybrid containing five

residues of RNA, but is

required for stability of short

DNA-RNA complex (BiaCore

experiments).

When the RNA grows beyond five or six residues, however, it must

compete with TFIIB for space on the Pol II saddle. If TFIIB wins the

competition, initiation is aborted and must be tried again. If the RNA

wins, TFIIB is ejected and Pol II is released from the promoter to

continue and complete transcription.

The B finger thus explains two crucial but for a long time mysterious

aspects of Pol II transcription, abortive initiation and promoter escape. In

these respects, it resembles the sigma factor in bacterial transcription.

21

Model of open promoter complex

Structure of an RNA polymerase II-TFIIB complex and the transcription

initiation mechanism

Science, 2010, Kornberg Lab


Transcription Initiation – TATA box binding protein (TBP)

Sigler, Burley, 1993

TBP forms a saddle shaped molecule with stirrups. A b-sheet in TBP forms

the DNA-binding site. TBP binds in the minor groove (strong hydrophobic

interaction, H-bonds) and induces large structural changes in DNA (DNA

helices on both sides form an angle of ca. 110º.

TBP: highly conserved CTD (180 aa); non-conserved NTD

24

Initiation: Model of an RNA polymerase II-TBP-TFIIB-DNA complex

Structure of the

C-terminal region

of TFIIB (pink)

complexed with

TBP (green) and

TATA-box

containing DNA

was docked to

the structure of

the Pol II-TFIIB

complex (clamp,

yellow), TFIIB-

NT-region), wall

(blue).

25

Model of an RNA polymerase II-TBP-TFIIB-DNA complex

after adding

straight B-form

DNA:

TATA-box-

saddle: 15bp;

saddle-active

site: 12 bp

= ca 27 bp!!

distance TATA-

box to

transcription start

site in promoters

25-30 bp


Docking a complex of a C-terminal TFIIB fragment, the TATA-binding

protein (TBP) subunit of TFID, and a TATA box DNA fragment:

First, the DNA fit snugly against the protein: – TBP evidently

configures promoter DNA to the contours of the Pol II surface.

Second, the DNA downstream of the TATA box ran past the saddle.

The distance from the TATA box to the saddle is about 1.5 turns of the

double helix, or 15 base pairs (bp).

We know from the transcribing complex structure that about 12

residues are required to cross the saddle to the active site. The

sum of 15 bp from the TATA box and 12 residues to the active site is 27

bp, closely coincident with the spacing of 25–30 bp from the TATA

box to the transcription start site of almost all Pol II promoters. In

this way, Pol II-TFIIB interaction may determine the location of the

transcription start site.


Initiation: Transcription bubble (complex with TFIIF)

The structure includes a complete

transcription bubble – not only the

template DNA strand with

associated RNA, but also the

nontemplate DNA strand, and the

region upstream of the bubble

where duplex DNA is reformed

following transcription.

yellow: TFIIF; green: coding DNA;

red: RNA; cyan: template DNA

The interaction of the

nontemplate strand with TFIIF

may trap a transient bubble in

promoter DNA, leading to the

initiation of transcription.


Transcription initation

The structures

of Pol II, TBP, and

TFIIB come from

X-ray

crystallography.

The structures of

TFIIE, TFIIF, and

TFIIH (helicase)

are from electron

crystallography

and from cryo-

electron

microscopy and

single particle

analysis.


Transcription initation - Complete minimal RNA

polymerase II transcription initiation complex

TBP bends the promoter DNA around

the polymerase and the CTD of TFIIB.

The NTD of TFIIB brings the DNA to a

point on the polymerase surface from

which it need only follow a straight path

and, by virtue of the conserved spacing

from TATA box to transcription start site

in Pol II promoters, the start site is

juxtaposed with the active center.

TFIIE enters the complex and recruits

TFIIH, whose ATPase/helicase

subunit introduces negative

superhelical tension in the DNA.


Transcription initation - Complete minimal RNA

polymerase II transcription initiation complex

Thermal unwinding produces a

transient bubble, which is captured

by TFIIF binding to the nontemplate

strand. The DNA can now bend in the

single stranded region and descend into

the Pol II active center.

Initiation and the synthesis of RNA

ensue, initially stabilized by the B finger.

Synthesis of a transcript greater than

about 6 residues in length leads to the

displacement of TFIIB, promoter

escape, and the completion of

transcription.



Other essential tasks of transcription:

Translocation

Nucleotide addition

Fidelity of Transcription

RNA escape

Regulation – the role of Mediator


Translocation: Bridge helix might serve as molecular ratched

Straight and bent states of the bridge helix in RNA polymerase II (yeast) and

bacterial RNA polymerase structures. The bend produces a movement of ≈3 Å in

the direction of the template strand, corresponding to one base pair step along

the strand.


A cycle of nucleotide addition by RNA polymerase II

At the upper left, the

structure of the

transcriping complex is

shown, omitting all but

the DNA and RNA near

the active center and

the bridge helix

(green).

The ribonucleotide in

the active center, just

added to the RNA

chain, is yellow.

At the lower left is the

structure after

translocation of DNA

and RNA across the

Pol II surface.



At the lower right is

the structure with an

unmatched NTP in

the entry (E) site. At

the upper right is

the structure with

NTP, matched for

pairing to the coding

base in the template

strand, in the

addition (A) site.



All four NTPs were

seen to bind an entry or

“E” site, whereas only

the NTP correctly

matched for base

pairing with the coding

base in the DNA was

seen to bind in the

active center, at the

nucleotide addition or

“A” site. The orientation

of NTP in the E site

was inverted with

respect to that in the A

site, leading to the

suggestion that NTPs

in the E site rotate to

sample base pairing in

the A site.


Bridge helix update

Cheung et al, Structural basis of initial RNA polymerase II transcription, EMBO

J, 2011


But 3D structure did not explain the fidelity of transcription: The

energy of base pairing, through two or three hydrogen bonds to the

template DNA, is far less than required to account for the selectivity of

the polymerase reaction.

2006:

New structures of RNA polymerase II (Pol II) transcribing complexes

reveal a likely key to transcription. The trigger loop swings beneath a

correct nucleoside triphosphate (NTP) in the nucleotide addition site,

closing off the active center, and forming an extensive network of

interactions with the NTP base, sugar, phosphates, and

additional Pol II residues. A His side chain in the trigger loop,

precisely positioned by these interactions, may literally “trigger”

phosphodiester bond formation. Recognition and catalysis are

thus coupled, ensuring the fidelity of transcription.


Fidelity of transcription: Trigger loop contacts NTP in the A site

Template DNA RNA

Trigger Loop

NTP in A site

(purine,

pyrimidine NT)

The trigger loop

swings beneath a

correct nucleoside

triphosphate

(NTP) in the

nucleotide addition

site, closing off the

active center, and

forming an

extensive

network of

interactions with

the NTP base,

sugar,

phosphates, and

additional Pol II

residues.


Fidelity of transcription: Trigger loop contacts NTP in the A site

Template

DNA RNA

Trigger

Loop

NTP in A site

(purine,

pyrimidine

NT)

A His side chain in

the trigger loop,

precisely positioned

by these

interactions, may

literally “trigger”

phosphodiester

bond formation.

Recognition and

catalysis are thus

coupled, ensuring

the fidelity of

transcription.


The trigger loop contacts all

moieties of the NTP - the base,

the phosphates and through other

Pol II residues, the sugar as well.

The resulting network of

interactions even includes the 2’-

OH group of the nucleotide just

added to the end of the RNA.

The importance of these interactions is shown by mutations affecting

transcription.


Trigger loop couples nucleotide selection to catalysis

Alignment of the trigger loop

with the NTP and the

precise positioning of a

histidine side chain, 3.5 Å

from the β-phosphate. The

histidine promotes the flow

of electrons during

nucleophilic attack of the 3′-

OH at the chain terminus

and phosphoanhydride bond

breakage. It serves as a

proton donor for the

pyrophosphate leaving

group. It literally triggers

phosphodiester bond

formation.

45

Nucleotide selection by alignment with the trigger

loop, coupling recognition to catalysis

The electronic transactions involved in trigger loop function require precise alignment of the

interacting moieties. This is achieved for a correct NTP by formation of the trigger loop

network. In the case of an incorrect NTP, for example a 2′-deoxy NTP, misalignment is

profound. A double helix formed with a 2′-deoxy nucleotide is 2 Å narrower than that formed

by a ribonucleotide.


Separation of RNA transcript from the template

- 3D structure in the posttranslocation state

Westover, K.D., et al. (2004) Structural basis of transcription:

separation of RNA from DNA by RNA polymerase II. Science.

-7

-8

-9

-10

Fork

loop

Rudder Lid

Release of RNA transcript

from DNA -RNA hybrid

revealed in the structure of

an RNA polymerase II

transcribing complex. The

upstream end of the DNA -

RNA hybrid helix, 7-10

residues from the active

center, is shown on the

left, with distances

between the DNA and RNA

bases indicated. The entire

DNA -RNA hybrid helix is

shown on the right, along

with protein loops involved in

helix melting (rudder and lid)

and stabilization (fork loop).






-7

-8

-9

-10

Fork

loop

Rudder Lid

Base pair 7 of the DNA-

RNA hybrid in this

structure appears normal

– the bases are

coplanar, with a distance

appropriate for hydrogen

bonding between them.

Base pairs 8, 9, and 10,

however, show

increasing deviations,

and consequent splaying

apart of the DNA and

RNA strands. The strand

separation is due to the

intervention of three

protein loops, termed

fork loop 1, rudder, and

lid.






-7

-8

-9

-10

Fork

loop

Rudder Lid

Rudder and lid lie

between DNA and RNA.

Rudder contacts DNA,

Lid RNA. A Phe side

chain of the lid serves as

wedge to maintain

separation of the strands.

Fork loop contacts the

sugar-phosphate

backbone of the hybrid

helix at base pairs 6 and

7, stabilizing the helix,

preventing the DNA-RNA

hybrid from unraveling

further and inhibiting

transcription.


Transcription

regulation:

the role of

Mediator

Mediator is a key

regulator of eukaryotic

transcription,

connecting activators

and repressors bound

to regulatory DNA

elements with Pol II.


Transcription regulation: the role of Mediator

Cryo-EM structure, 35 Å resolution, Asturias Lab

2002; Extension of the structure to atomic

resolution will one day reveal the regulatory

mechanism…

In the yeast Saccharomyces

cerevisiae, Mediator comprises

25 subunits with a total mass of

more than one megadalton and is

organized into three modules,

called head, middle/arm and tail.

Architecture of the Mediator head module, nature

2011; x-ray structure of mediator head; 4.3 A

51

Transcription regulation: the role of Mediator

In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a

total mass of more than one megadalton and is organized into three modules,

called head, middle/arm and tail.

Structure of the Mediator head module:

Laurent Larivière, et al, Nature, 492, 448–451, (20 December 2012); 3.4 Å

Documents

TRANSCRIPTION a) Transcription in prokaryotes b ...libvolume1.xyz/.../inhibitorsofprokaryotictranscriptiontutorial2.pdf2 Fig. Stryer Transcription in prokaryotes and eukaryotes The